Batas B, Chaudhuri J B
School of Chemical Engineering, University of Bath, Bath BA2 7AY, United Kingdom.
Biotechnol Bioeng. 1996 Apr 5;50(1):16-23. doi: 10.1002/(SICI)1097-0290(19960405)50:1<16::AID-BIT3>3.0.CO;2-4.
A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size-exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel-filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S-100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step.
已开发出一种在单次操作中提高重折叠产率并增加重折叠蛋白浓度的新方法。该方法使用尺寸排阻色谱基质进行缓冲液交换、聚集体去除和折叠反应。已证明蛋白质在凝胶过滤介质中扩散减少可抑制部分折叠分子的非特异性相互作用，从而减少聚集。使用Sephacryl S - 100（HR），成功从高达80 mg/mL的初始蛋白质浓度重折叠了鸡蛋清溶菌酶（HEWL）和牛碳酸酐酶（CAB）。溶菌酶的聚集反应减少，仅在所用最高蛋白质浓度下检测到。溶菌酶的平均回收率为63%，平均比活性为104%。碳酸酐酶实验也表明聚集受到抑制，从柱中回收的蛋白质平均为56%，比活性为81%。该过程能够在一步中实现活性物种的重折叠和纯化。
